Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature through an infectious cycle that alternates between mammals and a tick vector. Like many bacterial pathogens, B. burgdorferi must adapt to a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi has an unusual segmented genome that includes a large number of linear and circular plasmids. Increasing evidence indicates that plasmid-encoded genes are critical for successful adaptation by B. burgdorferi to the different environments that the spirochete encounters during its infectious cycle. A major focus of this project is to determine how and why the Lyme disease spirochete maintains such a unique genomic structure and the specific contributions of individual plasmids and genes at each stage of the infectious cycle. We have addressed this topic primarily through a molecular genetic approach. The complex genome of B. burgdorferi consists of a linear chromosome and more than 20 linear and circular plasmids. These plasmids encode numerous proteins critical to the spirochetes infectious cycle and many hypothetical proteins whose functions and requirements are unknown. In FY2009, Aaron Bestor and Dr. Phil Stewart adapted the Cre-lox recombination system in order to more efficiently manipulate the B. burgdorferi genome. The use of Cre to engineer large internal deletions within the chromosome or plasmids presents unique advantages relative to other available methods for genetic manipulation of B. burgdorferi, such as allelic exchange. The low transformation efficiency of B. burgdorferi precludes targeted inactivation of each individual gene as a practical means to investigate the role of every gene product in vivo. Therefore, we adapted the Cre-lox system for B. burgdorferi as an efficient tool with which to screen large segments of DNA in order to focus on loci relevant to fitness and survival in vivo. This system utilizes the Cre protein, a tyrosine site-specific DNA recombinase, and two short nucleotide recognition sequences called loxP. Sequential breaking and rejoining of loxP sites in a directly repeated orientation by the Cre recombinase results in the functional deletion of one site and the intervening target sequence. The Cre-lox system requires no accessory proteins or host co-factors and proceeds efficiently with both supercoiled and linear DNA. To test the feasibility of using the Cre-lox system in B. burgdorferi, Cre recombinase was introduced into a B. burgdorferi strain containing a shuttle vector encoding green fluorescent protein (GFP) with flanking loxP sites. As anticipated, Cre-mediated recombination resulted in excision and loss of gfp from the shuttle vector, which was observed by loss of fluorescence from the spirochetes;sequencing confirmed that gfp had been excised, leaving only a single loxP site. These results demonstrated that Cre functioned efficiently and could be used to engineer mutations in B. burgdorferi. After demonstrating that Cre/loxP functioned efficiently in B. burgdorferi, Aaron undertook a systematic deletion mapping of a large linear plasmid, lp54, which is present in all characterized B. burgdorferi isolates and stably maintained during in vitro propagation. Many of the hypothetical open reading frames (ORFs) on lp54 are regulated by the environmental conditions that distinguish the tick vector and mammalian host. Previous array studies indicated a higher percentage of temperature-regulated genes on lp54 than on the chromosome or any other plasmid in the genome. These traits suggest that a number of loci on lp54 are likely to contribute to the spirochetes survival throughout the infectious cycle. However, determining precisely which genes on lp54 are important is challenging because the majority of the 76 predicted ORFs encode proteins of unknown function. Therefore, Aaron used Cre-lox as a screening tool to delete large segments of lp54. He engineered the deletions in two parts, encompassing 14 genes on one half of lp54. The phenotypes of the resulting deletion mutants were assessed both in vitro and in vivo. All mutant strains grew comparably to wild type in liquid medium and were infectious for mice by needle inoculation, indicating that these conserved and highly regulated genes encompassing a large segment of lp54 are not critical to B. burgdorferis ability to infect and disseminate in a mammalian host. To complete the infectious cycle analysis, Aaron investigated the phenotype of the lp54 deletion mutants in ticks. Mutant spirochetes were acquired by feeding ticks and maintained at densities similar to that of wild-type B. burgdorferi. Subsequent feeding of the infected ticks resulted in infection of nave mice. We conclude that no sequences in the deleted region of lp54 are required by B. burgdorferi for tick acquisition, colonization or transmission. Considering the highly conserved and differentially expressed nature of lp54, we found it very surprising that the first 14 genes of the plasmid could be deleted without significantly affecting B. burgdorferis overall fitness at any stage of the infectious cycle. Little is known about this region of lp54, but previous studies have suggested possible contributions by one or more of these genes during the infectious cycle. Our data demonstrate that this region of lp54 does not encode any proteins or small regulatory RNAs that are required for mouse or tick infectivity. This study also demonstrates the efficiency and usefulness of the Cre-lox system as a screening tool with which to scan the B. burgdorferi genome for important loci. Although significant advances have been made in the genetic manipulation of B. burgdorferi, some simple molecular genetic techniques commonly applied to other bacteria are still not available. Beta-galactosidase, encoded by lacZ, has been used extensively as a convenient reporter gene in E. coli and is applicable to a broad range of organisms, both prokaryotic and eukaryotic. Beta-galactosidase activity can be monitored easily and quickly by simple color change assays for bacteria grown in both liquid and solid media, neither of which require expensive or specialized equipment. We were interested in determining if Beta-galactosidase assays could be used as a reporter to monitor gene expression in B. burgdorferi. Since the B. burgdorferi genome has a predominantly A-T rich sequence, Beth Hayes designed a synthetic lacZ gene that was optimized for the codon preference of B. burgdorferi (lacZ-Bb) and developed a shuttle vector containing lacZ-Bb, with and without a constitutive promoter. Beth found that only the B. burgdorferi colonies carrying the constitutively expressed lacZ-Bb gene change color in the presence of substrate. In addition, using a quantitative assay, Beth could measure Beta-galactosidase activity in B. burgdorferi carrying the constitutive LacZ-Bb construct, whereas only background levels were detected in B. burgdorferi containing the promoterless lacZ-Bb. These results demonstrate that an active Beta-galactosidase enzyme is made by B. burgdorferi when lacZ-Bb is expressed from a constitutive promoter. In order to confirm that lacZ-Bb could be used as an accurate reporter for transcriptional regulation of endogenous genes, Beth engineered fusions between lacZ-Bb and the promoters of two genes that vary in expression in B. burgdorferi at different stages of the infectious cycle. Introduction of these constructs into B. burgdorferi demonstrated that &#946;-galactosidase activity accurately reflects promoter strength and differential expression of endogenous genes in B. burgdorferi